BACKGROUND: Ultrasound (US) treatment was applied to increase the production of β-1,3-glucanase and chitinase by Beauveria bassiana (IBCB 66) cultivated in submerged fermentation. Application of US at different growth stages, US power outputs, time of ultrasonication, and duty cycles were the parameters evaluated. RESULTS: The best strategy comprised ultrasonication during 5 min at 24 h of fermentation with a power output of 195 W (fixed frequency of 24 kHz) and a duty cycle of 0.5 s.s−1 (0.5 s ON, 0.5 s OFF). The US treatment resulted in a production increase of 46% for β-1,3-glucanase and 42% for chitinase compared to the non-sonicated control. Fragmentations of the mycelia in the sonicated samples were observed in Scanning Electron Microscopy (SEM) micrographs after the US application, supporting the idea of a better release of enzymes to outside the cell. CONCLUSION: Ultrasound-assisted fermentation was a promising tool to increase the production of β-1,3-glucanase and chitinase by Beauveria bassiana (IBCB 66). The combination of US parameters showed to be decisive in obtaining higher enzyme production when compared to the control non-sonicated experiment. Therefore, this work brings a significant contribution to the field of stimulation of bioprocesses with ultrasound since it shows a strategy to obtain higher enzyme production when compared to traditional fermentation. © 2020 Society of Chemical Industry.